EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription.

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress.

Dynamics of the membrane-cytoskeleton interface in MHC class II-restricted antigen presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

Kinesin KIFC1 actively transports bare double-stranded DNA.

During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport. Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA. Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

T-cell artificial focal triggering tools: linking surface interactions with cell response.

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.

Myosin V stepping mechanism.

We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V.

Driven lattice gas of dimers coupled to a bulk reservoir.

We investigate the nonequilibrium steady state of a one-dimensional (1D) lattice gas of dimers. The dynamics is described by a totally asymmetric exclusion process (TASEP) supplemented by attachment and detachment processes, mimicking chemical equilibrium of the 1D driven transport with the bulk reservoir. The steady-state phase diagram and current and density profiles are calculated using both a refined mean-field theory and extensive stochastic simulations. As a consequence of the on-off kinetics, a phase coexistence region arises intervening between low and high density phases such that the discontinuous transition line of the TASEP splits into two continuous ones. The results of the mean-field theory and simulations are found to coincide. We show that the physical picture obtained in the corresponding lattice gas model with monomers is robust, in the sense that the phase diagram changes quantitatively, but the topology remains unaltered. The mechanism for phase separation is identified as generic for a wide class of driven 1D lattice gases.

Dynamic correlation functions and Boltzmann-Langevin approach for driven one-dimensional lattice gas.

We study the dynamics of the totally asymmetric exclusion process with open boundaries by phenomenological theories complemented by extensive Monte Carlo simulations. Upon combining domain wall theory with a kinetic approach known as Boltzmann-Langevin theory we are able to give a complete qualitative picture of the dynamics in the low- and high-density regimes and at the corresponding phase boundary. At the coexistence line between high- and low-density phases we observe a time scale separation between local density fluctuations and collective domain wall motion, which are well accounted for by the Boltzmann-Langevin and domain wall theory, respectively. We present Monte Carlo data for the correlation functions and power spectra in the full parameter range of the model.